1. Heparan Sulfate Proteoglycans Regulate Fgf Signaling and Cell Polarity during Collective Cell Migration 
Marina Venero Galanternik, Kenneth L. Kramer, Tatjana Piotrowski 
Cell Reports 
Volume 10, Issue 3, Pages (January 2015)
DOI: /j.celrep
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2. Cell Reports 2015 10, 414-428DOI: (10.1016/j.celrep.2014.12.043)
Copyright © 2015 The Authors Terms and Conditions

3. Figure 1
Loss of HSPGs Results in LL Prim Migration and Patterning Defects
(A, E, I, and M) gpc1b expression in (A) WT, (E) apcmcr, (I) DMSO-treated, and (M) SU5402-treated embryos.
(B, C, F, G, J, K, N, and O) gpc4 and sdc3 expression in (B and C) WT, (F and G) apcmcr, (J and K) DMSO-treated, and (N and O) SU5402-treated embryos.
(D, H, L, and P) sdc4 expression in (D) WT, (H) apcmcr, (L) DMSO-treated, and (P) SU5402-treated embryos.
(G and H) Black arrowheads point to the ectopic halo of sdc3 and sdc4.
(Q and R) WT and extl3 mutant prim has reached the tail tip by 48 hpf.
(S and T) ext2 and extl3/ext2 prim fails to complete migration.
(Q’–T’) Magnification of prim at 46 hpf.
(Q’ and R’) WT and extl3 mutant prim shows a normal morphology.
(S’) ext2 mutant prim possesses a pointed tip (white arrow).
(T’) extl3/ext2 mutant prim shows severe morphological defects. The leading edge elongates (white arrow) and rosettes are closely spaced (yellow arrowheads).
(U–W) Still images of time-lapse movies: WT (U), ext2 (V), and extl3/ext2 mutant prim (W). Yellow arrows in (V) and (W) point to ectopic filopodia.
See also Figure S2 and Movies S1, S2, S3, and S4.
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4. Figure 2
The Wnt/β-Catenin Pathway Is Ectopically Activated in extl3/ext2 Mutants
(A–M’) In extl3/ext2 mutants Wnt/β-catenin target genes expand progressively between 34 and 48 hpf.
(F, H, J, and L) Sibling prim migration has finished by 48 hpf.
(G, I, K, and M) In extl3/ext2 mutants, the prim stalls and lef1, axin2, sef, and fgf10 are upregulated.
(G’, I’, K’, and M’) Magnified views of extl3/ext2 mutant prim. Black arrows: Wnt/β-catenin target expression in interneuromast cells.
(N–U’) Time course of the Wnt/β-catenin target wnt10a and inhibitor dkk1b. Upregulation of wnt10a correlates with the downregulation of dkk1b.
(O, O’, S, and S’) Mild downregulation of dkk1b corresponds with the expansion of wnt10a at 40–42 hpf.
(P, P’, T, and T’) By 44–46 hpf, wnt10a expands along the prim and dkk1b expression is almost lost.
(Q, Q’, U, and U’) By 48–50 hpf, dkk1b expression is entirely reduced, resulting in complete expansion of wnt10a.
(V–Y’’) The Fgf target pea3 is gradually lost in the prim (V–Y’) but is progressively activated in surrounding cells (V’’–Y’’).
(Z and Z’) Lower-magnification images of the 44–46 hpf embryos in X, X’, and X’’.
See also Figures S2 and S3.
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5. Figure 3
Fgf Signal Transduction Is Progressively Lost in extl3/ext2 Mutant Prim
(A–B’) ZO-1 staining reveals apical constrictions (white arrows) in 48 hpf extl3/ext2 neuromasts and prim rosettes, but not in interneuromast clumps (yellow arrow).
(C–D’) LL hair cells precursors are present in 48 hpf extl3/ext2 mutants, as evidenced by atoh1a and s100t expression. Black arrows in (C), (D), and (D’) indicate the position of the prim.
(E–H’’) Dp-ERK staining reveals remaining low levels of Fgf signaling in the 48 hpf prim. Dp-ERK is progressively downregulated as rosettes collapse (E’’–H’’). By 5 dpf, no protein remains inside the prim (H’).
(I–J’) By 3 dpf, most of the formed hair cells die inside the collapsing neuromast.
(I’ and J’) Neuromasts outlined in the white boxes in (I) and (J).
(K–L’) Inhibition of Fgf signaling accelerates the onset of the extl3/ext2 phenotype. extl3/ext2 mutants and their siblings treated with SU5402 from 28 to 34 hpf show complete upregulation of the Wnt target lef1.
See also Figures S2, S3, and S5 and Movie S4.
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6. Figure 4
NaClO3 Treatment Phenocopies extl3/ext2 Mutants, Causing Loss of Fgf Signaling and Resulting in Expansion of the Wnt Domain
(A and B) In NaClO3-treated embryos, the prim stalls.
(C–D’’) NaClO3-treated embryos show downregulation of HS levels by 10E4 antibody staining.
(E–F’’) extl3/ext2 mutants show a similar loss of HS.
(G–H’) NaClO3-treated Wnt reporter line embryos show expansion of the Wnt domain in the prim, as evidenced by destabilized GFP (dGFP) expression and dgfp in situ hybridization.
(I–J’) The Fgf target pea3 is restored in NaClO3-treated prim after CAfgfr1 induction.
(K–L’) Restored Fgf signaling restricts expression of the Wnt target lef1 back to the leading region.
(M–N’) The restored restriction correlates with the activation of dkk1b after Fgf pathway activation.
(O–T’) Wnt is restricted after NaClO3 treatment by inducing Dkk1b expression in the Tg(hsp70:dkk1b-GFP) line. In the absence of HS, Dkk1b induction is sufficient to restrict the Wnt targets lef1 and fgf3 back to the leading region (O–P’ and Q–R’), but not to restore Fgf signaling in the trailing region of the prim (S–T’). pea3 is still only expressed in cells surrounding the prim (T’).
See also Figure S4, Table S1, and Movies S5 and S7.
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7. Figure 5
Chemokine Receptor Expression Correlates with Prim Stalling in extl3/ext2 Mutants
(A–H) cxcr4b time course in 32–50 hpf siblings and extl3/ext2 mutants. cxcr4b is upregulated at 32–34 hpf and increases as the embryo develops.
(I–P) cxcr7b is expressed normally between 32 and 42 hpf in extl3/ext2 mutants, but becomes downregulated at 44 hpf until it is absent by 50 hpf. These expression changes correlate with the expansion of Wnt signaling (Figure 2) and slowing down and stalling of the prim.
(Q–X) cxcl12a time course shows that despite the upregulation of cxcl12a at 32 hpf, the prim continues to migrate until cxcr7b is downregulated by 44–46 hpf (M–P). cxcl12a upregulation continues as the extl3/ext2 phenotype becomes severe by 50 hpf (W and X).
(Y, Y’, and Z) extl3/ext2 mutants possess more Engrailed (4D9)-positive medial fast muscle fibers along the trunk. In (Z) the SE is indicated.
See also Figure S4.
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8. Figure 6
Loss of Fgf Signaling and HS Function Leads to Random, Dynamic Filopodia Formation
(A–B’) BIO-treated Tg(claudinB:GFP) embryos show upregulation of cxcl12a compared to control embryos, but no evidence of ectopic filopodia formation. BIO treatment leads to upregulation of the Wnt and Fgf pathways in the prim.
(C–D’) In NaClO3-treated Tg(claudinB:GFP) embryos where cxcl12a is upregulated. Filopodia form along all LL cells (D’). Similarly to extl3/ext2 mutants, these embryos show upregulation of Wnt and loss of Fgf signaling.
(E–F’) cxcl12a is only slightly upregulated in ext2 mutants. Mild ectopic filopodia are observed in the most anterior region of the prim by time-lapse recording (F’; Movie S3); 48 hpf ext2 mutants show only partial Wnt upregulation and Fgf signaling downregulation (data not shown).
(G–H’) Bmp signaling inhibition with Dorsomorphin leads to an increase in cxcl12a-expressing muscle cells. Bmp inhibition does not alter prim migration or signaling, but induces the upregulation of cxcl12a.
(I–J’) Inhibition of Fgf signaling by SU5402 causes the formation of ectopic filopodia, but not upregulation of cxcl12a (J’). Wnt is upregulated as a result of Fgf inhibition.
(K and L) Inhibition of Fgf signaling in extl3/ext2 mutants does not eliminate ectopic protrusions. Black arrows point to ectopic filopodia.
See also Figures S4 and S5 and Movies S6 and S8.
Cell Reports  , DOI: ( /j.celrep )
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9. Figure 7
HSPGs Control Cell Polarity, Ligand Distribution, and Activation of Wnt and Fgf Signaling during Collective Cell Migration
(A) HSPGs are expressed in discrete domains of the LL prim and their expression is dependent on Wnt or Fgf signaling, making them part of a feedback loop. Fgf-dependent ERK signaling surrounds the border of the prim.
(B) Analysis of HS-depleted embryos (extl3/ext2 mutants or NaClO3-treated) reveals that HSPGs are important for localized Wnt and Fgf signaling activation, as well as for limiting diffusion of Fgf ligands away from the prim. In the absence of HS, Fgf ligands activate Fgf signaling outside of the prim.
(C) Constitutive activation of Fgfr1 rescues Fgf signaling in HS-depleted embryos, demonstrating that Fgf requires the presence of HS for Fgf signal transduction.
(D) Induction of Dkk1b in HS-depleted embryos is sufficient to restrict Wnt back to the leading region, demonstrating that Wnt expands as a result of Fgf signaling loss. Dkk1b induction is not sufficient to rescue Fgf signaling, because HS are required for Fgf signal transduction in the prim. Additionally, the Fgf-dependent pea3 halo is still present after CAfgfr1 and Dkk1b rescue experiments, as HS plays an important role in limiting Fgf diffusion.
(E) HS also controls the cell polarity of LL prim cells, as HS-depleted embryos extend dynamic ectopic filopodia.
Cell Reports  , DOI: ( /j.celrep )
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